Lens membrane preparations have been shown to have a remarkable rigidity which increases in the inner nuclear region of the lens and has been correlated with the cholesterol (C)/phospholipid (PL) ratio. However, the distribution of these lipids in single lenses had not been determined. Utilizing a new technique for isolating consecutive layers of a human lens, lipid composition and contents of seven pairs of normal lenses from subjects ranging from 54 to 77 years old have been analyzed. It was found that the PL content remains relatively constant at 22-24 micrograms/mg through all but the nuclear 10-15% of the lens dry weight where it drops precipitously to about 7 micrograms/mg. The C distribution is more complex; the C content is at a low level of 14 micrograms/mg in the outer cortical 15-20%, rises to 25 micrograms/mg in the inner cortical 40-60% of the total lens weight, and drops to 12 micrograms/mg upon reaching the nucleus. Thus, the continuous increase in the lens C/PL ratio is due to the increase in C in the cortex and the large decrease in PL in the nucleus. Analyses of phospholipid and fatty acid composition in the different regions of the lens indicate significant differences. However, the abundance of mono-unsaturated fatty acids contributing to the rigidity of the membrane has only minor variation. The lens has a remarkably low overall lipid content of 4% and only 2% in the nuclear region. Calculation of the surface area of the nuclear fiber cell suggests that less than one-third of the membrane is made of PL bilayer. Thus, a mosaic of PL and C patches or some other type of structure involving membrane fusion must be present. Conversion of the % dry weight occupied by the concentric fiber fractions to their location on the lens axis in mm indicates that the nuclear 15% dry weight of the tissue occupies more than 50% of the axial length. This region contains the embryonic lens and the primary lens fibers. Similarly, the metabolically active outer 20% of the dry weight accounts for less than 10% of the visual axial length and contains cells undergoing terminal differentiation. Cataractous lenses have lipid distributions similar to those of the normal lenses suggesting that membrane lipid is either not involved in cataract formation or that the primary insult is localized in an undetectable small number of fiber cells.